﻿A new toad of Oreolalax Myers & Leviton, 1962 (Anura, Megophryidae) from Sichuan Province, southwest China

﻿Abstract A new species of the genus Oreolalax Myers & Leviton, 1962 is described from Sichuan Province, southwest China. Molecular phylogenetic analyses based on mitochondrial gene sequences clustered the new species as an independent clade nested with O.rugosus, O.liangbeiensis, and O.major. The new species could be distinguished from its congeners by a combination of the following characters: body size moderate (39.8–52.8 mm in male); head broad; tympanum absent; interorbital region with dark triangular pattern; 1/3 toes webbed, with broad lateral fringes, belly smooth, brown yellow or medium yellow scattered variable brown spots; skin on dorsum relatively rough with fine tiny and large warts granules; middle pectoral glands are evident in males; flanks with dark-brown warts granules; upper surface of limbs with dark bars; and iris orange above and creamy-white below. The new species inhabits subtropical alpine scrub and swamp.

Hengduan Mountains, located in the southeastern part of Qinghai-Tibet Plateau, have a complex terrain and significant vertical climate changes.The mountain ecosystem in this region boasts a diverse range of habitats, capturing global attention for its biodiversity, and its speciation and protection have been of concern.During the field surveys in 2023 in Yanyuan City, Sichuan Province (Prov.),southeastern Hengduan Mountains, China, we collected eight adult and seven tadpole Oreolalax specimens.Our detailed morphological comparisons and molecular phylogenetic analyses indicated that these specimens should represent an undescribed species.Herein we describe it as a new species.

Sample
Through a field survey in June 2023, a total of 15 samples of the undescribed species including eight adult males and seven tadpoles were collected nocturnally from Shuhe town, Yanyuan County, Sichuan Province, China (Suppl.material 1; Fig. 1).Taxonomic assignments of tadpoles were confirmed by molecular results.After taking photographs, the toads and tadpoles were euthanized using isoflurane, and the specimens were fixed and preserved in 75% ethanol.Tissue samples were taken and preserved separately in 95% ethanol prior to fixation.Specimens were deposited in Chengdu Institute of Biology, Chinese Academy of Sciences (CIB, CAS).

Molecular phylogenetic analyses
Genomic DNA from each specimen collected in this work was extracted using a TIANamp Genomic DNA Kit by TIANGEN (BEIJING) BIOTECH, China.Fragments of the mitochondrial genes 16S rRNA and cytochrome c oxidase I (COI) genes were amplified.Primer sequences were retrieved from the literature for 16S (Simon et al. 1994) andCOI (Che et al. 2012).PCR amplifications for the 16S/COI gene were performed in a 25 mL volume reaction with the following conditions: an initial denaturing step at 95 °C for 4 min; 36 cycles of denaturing at 95 °C for 40 s, annealing at 55 °C/50 °C for 40 s and extending at 72 °C for 70 s, and a final extending step of 72 °C for 10 min.Sequencing was conducted using an ABI3730 automated DNA sequencer at Sangon Biotechnologies Co., Ltd.(Shanghai, China).
Sequences were assembled and aligned using Mega v. 7.0 (Kumar et al. 2016) A total of 1084 bp (16S+COI gene) sequences were obtained from the Oreolalax species.The maximum likelihood method (ML) and Bayesian inference (BI) method were used to construct gene trees to analyze the intraspecific phylogenetic relationship of species using PhyloSuite v. 1.2.2 (Zhang et al. 2020).
The best substitution models were inferred by the corrected Akaike information criterion (AICc) using the ModelFinder module in PhyloSuite software (BI: (GTR+I+G model for all partition); IQ: (GTR+F+R2: 16S, TPM2u+F+I+G4: COI)); in the ML analyses, we performed 1000 ultrafast bootstrap replicates based on the IQ-TREE.In the BI analyses, two runs each with four Markov chains were run for 20 million iterations with sampling every 1000 generations.The first 25% of generations were removed at the "burn-in" stage followed by calculation of Bayesian posterior probabilities and the 50% majority-rule consensus of the post burn-in trees sampled at stationarity.Trees were visualized with the Fig-Tree v. 1.4.2 program (Rambaut 2016).Finally, genetic distance was calculated with the pairwise uncorrected p-distance model between Oreolalax species on the COI gene using MEGA v. 7 (Kumar et al. 2016).

Morphological analyses
Measurements were made with a digital caliper to the nearest 0.1 mm.The terminology and methods followed Fei et al. (2009) and Watters et al. (2016).Twenty-three morphometric characters were measured for adults: SVL (snoutvent length), direct line distance from tip of snout to posterior margin of vent; HW (head width), at the widest point of the jaws angle; IOD (interorbital distance), the shortest distance between the anterior corners of the orbits; HL (head length), from the posterior of the jaws to the tip of the snout; ED (eye diameter), horizontally from the anterior to posterior corner of the eye; SL (snout length), distance from the tip of the snout to the anterior corner of the eye; UEW (upper eyelid width), greatest width of the upper eyelid margins, measured perpendicular to the anterior-posterior axis; IND (internarial distance), shortest distance between the inner margins of the nostrils; EN (eye-nostril distance), from anterior corner of the eye to the posterior margin of the nostril; NS (snout-nostril length), distance from the center of the external nares to the tip of the snout; LAHL (length of lower arm and hand), the length from the elbow to the end of the third finger; FAW (forearm width), greatest width of the forearm; THL (thigh length), distance from the vent to the knee; TL (tibia length), distance from the outer surface of the flexed knee to the heel/tibiotarsal inflection; TW (tibia width), maximum width of tibia along its length; FL (foot length), from the base of the inner metatarsal tubercle to the tip of Toe IV; LFT (length of foot and tarsus), the length from the tibial appendicular joint to the end of the fourth toe; Toe4L (toe IV length), from the metatarsal tubercle to the tip of Toe IV; Fin1L (finger I length), from the proximal edge of the palmar tubercle to the tip of the Finger I; Fin3L (finger III length), from the proximal edge of the palmar tubercle to the tip of the Finger III; IMT (inner metatarsal tubercle length), the greatest length of the inner metatarsal tubercle; IPTL (inner palmar tubercle length), maximum length of the inner palmar tubercle; OPTL (outer palmar tubercle length), maximum length of the outer palmar tubercle, measured parallel along forearm axis.Thirteen morphometric characters were measured for tadpoles: BH (maximum body height); BW (maximum body width); SVL (snout-vent length); MW (mouth width), distance between two corners of mouth; SL (snout length), distance from the tip of the snout to the anterior corner of the eye; SS (snout to spiraculum), distance from spiraculum to the tip of the snout; ED (maximum eye diameter); IND (internasal distance),minimum distance between nostrils; IOD (interocular distance), minimum distance between eyes; TAH (tail height), maximum height between upper and lower edges of tail; TAL (tail length), distance from base of vent to the tip of tail; TBW (maximum width of tail base); TOL (total length), distance from the tip of the snout to the tip of tail.

Molecular phylogenetic analyses
Phylogenetic results based on 16S and COI genes showed that the topological structures obtained by BI and ML analyses resulted in essentially identical topologies (Fig. 2).All samples of the undescribed species occupied an independent monophyly and were closely related to O. rugosus, O. major, and O. liangbeiensis.Genetic distances on the COI gene between all samples of the undescribed species were 0.0%-0.3%.The undescribed species is closest to O. rugosus on genetic distance (4.5%), being higher than, or at the same level, as many pairs of substantial species, such as O. liangbeiensis vs O. major (3.8%),O. liangbeiensis vs O. rugosus (3.3%) (Suppl.material 2).
The new species differs from its congeners by a combination of the following characters: body size moderate 39.8-52.8mm in male; head broad; tympanum hidden; distinct black spots present on flanks; toes 1/3 webbed, with broad lateral fringes; dorsal body deep brown or yellowish-brown; belly smooth, middle yellow, scattered fine mottling, abdominal margin more spotted; skin on dorsum rough with dense and varied size granules, warts are covered with dark spots or no spots; iris light orange or light yellow above, creamy silver white bellow; and middle pectoral glands are evident in males.
Hindlimbs relatively long, length 177% of body length; shank length subequal to thigh length, slightly shorter than foot length (THL 23.4 mm, TL 23.8 mm, FL 24.4 mm); heels partially overlap when thighs are positioned at right angles to the body and tibia-tarsal articulation reaches the middle eye when leg stretched; toes 1/3 webbed with distinct fringes; inner metatarsal tubercle long oval, small.
In life, dorsal body and head rough, back with moderate sparse granules, relatively small warts on head; with dark-brown triangular between eyes; dorsal arms and hindlimbs with small granules and bumps; distinct warts cover the fold and posterior of snout.Ventral skin smooth; pectoral glands flat; pectoral glands evident, chest spines and finger spines not visible; femoral glands slightly swollen, distinct on posterior thigh.Small verrucous granules around the anus.
Large brown markings on dorsum, dark brown triangular pattern between eyes; large markings dorsum brown; ventral skin medium yellow, with scattered little dark speckling; supratympanic fold dark brown; lateral head and flanks brown with dark patches; throat mixed pink and orange yellow, margin with small beige warts; chest pink and the pectoral glands medium yellow; forelimbs covered with black irregular spots; dorsal limbs yellowish-brown, the spots and stripes of dorsal upper arms and tibiotarsal articulation black; ventral arms, thigh, tibia medium yellow with flesh marking; upper iris light orange yellow, lower iris creamy white, both parts embedded black mesh lines.
In preservative (75% ethanol), dorsal body and head dark grey; irregular spots in forelimbs, black longitudinal stripes on hindlimbs; ventral surface beige, throat and arms beige white; with grayish-brown speckling; mandibular margin warts white; pectoral glands and ventral of the hindlimb beige yellow, scattered black spots; hand and feet dark grey, finger tips and palms grayish-white, inner metatarsal tubercle grey; lateral grey on snout and undereye, patches black; skins beneath supratympanic fold dark grey, flanks grey, covered with creamy white warts, black spots around the edges of warts.Perianal warts and femoral gland creamy-white (Fig. 4).
Tadpoles.Measurements see Suppl.material 4. Description based on preserved tadpole CIBSH20230603kd01 at Gosner stage 37 (TOL 62.3 mm, BL 20.6 mm) (Fig. 5).The mouth is located below the rostral end; labial tooth row formula I:(5+5) /(5+5):I; the upper lip papillae is large, the central missing length is close to 3 papillae position, the lower lip papillae is small and pointed; and there are small teeth on the auxiliary processes of the oral corner; jaw sheaths strong, serrated, the lip teeth are daggerlike; body elliptical in dorsal view, body width is 113% of height; snout rounded, eye positioned dorsolateral; SL 29% of BL; eyes relatively small, ED 8.3% of BL; nostrils near oval; tail long and muscled, TAL 210% of BL; TAH 113% of BH; TBW 44% of BW; SS 53% of BL.Body dark brown in the back and lateral view, creamy yellow in the ventral; tail brown; the single opening of the spiracle lateral, without a free distal tube; tail end blunt; faint brown cloud spots faintly visible on upper caudal fin, caudal fin light and broad.
By having dark bars on the limbs, the new species can differ from O. rugosus (vs.no or irregular), O. xiangchengensis, O. pingii, and O. puxiongensis (vs. no).
Oreolalax yanyuanensis is genetically closest to O. rugosus, O. liangbeiensis and O. major.In addition to the morphological differences (Fig. 6; Suppl.material 5), the new species distinctly differs from these three in measurement proportions.The new species can differ from O. rugosus by having larger LFT, TL, and smaller IN, IOD, UEW, TW.The new species distinctly differs from O. liangbeiensis by having larger HL, HW, ED, LAHL, TL, LFT and smaller SL, IOD.The new species distinctly differs from O. major by having larger HW, ED, TL, LFT and smaller SL, IN, IOD (Table 2).Etymology.The specific epithet "yanyuan" refers to the type locality of the species, Yanyuan County, Sichuan Province.We suggested the common name as "Yanyuan toothed toad", and the Chinese name as "Yan Yuan Chi Chan (盐源齿蟾)".

Discussion
Hengduan Mountains is part of the hotspot defined as "Mountains of Southwest China", and its biodiversity conservation has attracted much attention (CEPF 2024).At present, most Oreolalax species are distributed in the eastern Hengduan Mountains and the surrounding mountains of the Sichuan Basin at an altitude of 700-3550 m.More than half of Oreolalax species is listed as "threatened" by IUCN due to habitat loss, habitat degradation, logging, tourism development, and invasive species (Fei and Ye 2016;Jiang et al. 2016;IUCN 2024).Twelve species of Oreolalax are listed as vulnerable, near threatened, endangered, or critically endangered (IUCN 2024).Furthermore, some species have only been recorded at their type locality, and their habitat is not covered by any biodiversity conservation network, even with the high extinction risk (e.g., O. puxiongensis).Also, O. longmenmontis, which has not yet been evaluated by IUCN, faces low population and habitat loss (Hou et al. 2020).Assessments of habitat status, breeding activity, population size and dynamics are needed for these groups, especially for the newly discovered (e.g., O. yanyuanensis sp.nov.) and data deficient species (e.g., O. weigoldi).
The auditory system is critical for animals' survival and reproduction.Studies have shown that the thin air and low air density at high altitudes lead to slow sound speed, and animals singing in anoxic environment at high altitudes will consume a lot of energy, posing a threat to survival (Liao and Liu 2008;Wen 2014).Some amphibians have reduced investment in acoustic communication, resulting in structural degradation of acoustic communication organs which may be adapted to the high-altitude environment (Lehr and Trueb 2007;Wen 2014).In Oreolalax, the tympanic membranes are hidden or absent in most groups except O. rhodostigmatus.Some high-altitude groups (>3000 m), such as O. rugosus and O. xiangchengensis, showed the absence of columella, while the low-altitude groups showed a developed columella (Wei et al. 2009).Oreolalax yanyuanensis is distributed at high altitudes (3000-3200 m), but it is not clear whether the ear structure is degraded.Due to the limited number of specimens, we will use Micro-CT scans to explore the middle ear structure in the future.
In June 2023, the individuals of O. yanyuanensis found in the wild apparently lacked finger spines and chest spines, and no female individuals have been collected, so it is possible that its breeding season had ended.Based on its morphological characteristics, number and size of tadpoles, it is speculated that O. yanyuanensis may reproduce in April or May.Further studies are needed to investigate its reproductive behavior and population dynamics.

Conclusions
Based on morphological and molecular evidence, we revealed a new toad belonging to the Oreolalax species group-O.yanyuanensis sp.nov.The new species is so far only known from Shuhe town, Yanyuan County, south Sichuan Prov., China.The findings in this study improve our understanding of species diversity in the genus Oreolalax.More studies are necessary to uncover the population size, reproductive ecology, and habitat status to better protect the new Oreolalax species.

Figure 3 .
Figure 3. Photos of the holotype CIBSH20230603020 of Oreolalax yanyuanensis sp.nov.A dorsal view B ventral view; the white arrow indicates the position of the pectoral gland C lateral view D ventral view of hand E ventral view of foot.
material 1 (unit in mm).Half of the individuals had faint markings on the abdomen Dorsal markings are obvious in most individuals (CIBSH20230603016-22), except one (CIBSH20230603023).Lateral markings are obvious in most individuals (CIBSH20230603016-21, CIBSH20230603023), except one (CIBSH20230603022).Color of warts covering the back was yellowish-brown (CIBSH20230603022), light brown

Figure 4 .
Figure 4.The holotype CIBSH20230603020 of Oreolalax yanyuanensis sp.nov. in preservative A dorsal view B ventral view C lateral view D dorsal view of the left hand E ventral view of the left foot.
Figure 5. Photos for the tadpole CIBSH20230603kd01 in preservative of Oreolalax yanyuanensis sp.nov.A dorsal view B ventral view C lateral view D ventral view of head.

Figure 6 .
Figure 6.Specimen photos of Oreolalax yanyuanensis sp.nov.and its relative species A, B dorsal and ventral view of the holotype CIB25333 of O. rugosus C, D dorsal and ventral view of the topotype CIB24678 of O. liangbeiensis E, F dorsal and ventral view of the topotype CIB24695 of O. major G, H dorsal and ventral view of the topotype CIBSH20230603020 of Oreolalax yanyuanensis sp.nov.Scale bar: equal to 10 mm.

Table 1 .
Information for samples used in molecular phylogenetic analyses in this study."-" represents a lack of data.

Table 2 .
Morphometric comparisons between Oreolalax yanyuanensis sp.nov.and its relatives.Shaded values represent a ratio of body measurements to SVL that differs between these three species and the new species.